D-Peptide Inhibitors of HIV Assembly and Maturation Background: New therapies less susceptible to drug resistance are needed to combat the global HIV epidemic. Virus assembly and maturation remains a significant yet largely unexploited antiviral target. HIV assembly and maturation is mediated at the protein level by self-association of the HIV Gag polyprotein and of its sub-domains such as the matrix protein (MA) and capsid protein (CA). Peptides derived from MA and CA or selected from phage libraries have been shown to be able to block virus assembly and maturation in vitro. However, the antiviral effects of these peptide-based assembly/maturation inhibitors vary, due to a large extent to their poor stability in the protease-rich environment of the living cell - an inherent drawback of peptide therapeutics. Peptides composed entirely of D-amino acids, i.e., D-peptides, are resistant to proteolysis, which translates into much-improved bioavailability and reduced immunogenicity as compared with conventional L- peptides. Thus, D-peptides are ideally suited as lead drug compounds for therapeutic development. Objective/Hypothesis: We seek to develop, by the means of chemical protein synthesis and mirror-image phage display, D-peptide based antagonists of HIV MA and the C-terminal domain of CA (C-CA) as a novel class of therapeutic agents for the treatment of HIV-1 infection. Specific Aims: (1) Synthesize the D- enantiomers of HIV-1 MA and C-CA composed entirely of D-amino acids using a combination of solid phase peptide synthesis and native chemical ligation;(2) Screen phage-expressed peptide libraries against DMA and DC-CA and identify optimal sequences required for productive binding to the D-proteins;(3) Test the hypothesis that D-peptide antagonists of native MA and C-CA inhibit HIV replication in peripheral blood mononuclear cells. Study design: We will screen phage-expressed peptide libraries against chemically synthesized DMA and DC-CA, identifying high-affinity binders for the D-proteins. Inversion from L to D through peptide synthesis will convert phage-optimized L-peptide ligands to their mirror image D-forms that specifically bind native LMA and LC-CA. We will structurally characterize the interaction between the D-peptides and LMA or LC-CA, and evaluate their antiviral activity in vitro. In addition, mechanistic studies will be carried out to examine how the D-peptides disrupt HIV Gag assembly. Finally, novel delivery vehicles will be designed to improve peptide uptake by target cells. Significance: The proposed research seeks to fill the obvious gap by developing D-peptide inhibitors to prevent assembly of both immature and mature HIV particles. The specific aims outlined in this proposal, if achieved, will enable us to design D-peptide antiviral therapeutics for preclinical studies. This novel class of peptide antagonists, emulating the activity of known assembly/maturation inhibitors, enjoys high specificity and less toxicity compared with traditional small- molecule drugs, and is superior to conventional L-peptide-based antiviral agents with respect to bioavailability, pharmacokinetics, and immunogenicity. Identification of D-peptide antagonists may help better understand the virus assembly and maturation process and illuminate insight as well into designing other classes of antagonistic molecules to inhibit HIV replication. PUBLIC HEALTH RELEVANCE: Current antiretroviral therapy (ART) for HIV-1 infected patients utilizes a combination of inhibitors that target the viral enzymes reverse transcriptase and protease. ART reduces viral load and slows the progression of HIV to AIDS, contributing to a steady decrease in AIDS deaths in the world. Despite its success, ART does not eradicate HIV from infected cells, and, among many complications of ART is the emergence of drug-resistant HIV strains not responding to current antiretroviral regimens. The proposed research aims to develop a novel class of D-peptide inhibitors as additional weapons in the arsenal to fight HIV infection by specifically targeting HIV assembly and maturation.